The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferatoractivated receptor ␥ (PPAR␥) is required for this effect, at least in part due to direct activation of the PPAR␥1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBP␣ promoter, which exerts positive feedback on C/EBP␦ expression. Despite this, C/EBP␣ is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBP␤ and ␦, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2.The last decade has seen an enormous surge of interest in the biology of adipocytes, including the developmental processes by which these cells are formed. This has been fuelled by the convergence of a worldwide epidemic of obesity and diabetes (26, 37), with the recent realization that adipose tissue is an active secretory organ regulating a wide array of physiological processes.Adipogenesis represents a complex series of transcriptional events through which multipotent mesenchymal precursor cells become committed to the adipocyte lineage and ultimately express all of the genes typical of mature fat cells (31). These transcriptional events integrate a variety of extracellular signals that direct fat cell formation in time and space. Most of our knowledge concerning the transcriptional events mediating adipogenesis has come from cultured cell lines such as 3T3-L1 and 3T3-F442A (16, 17) that can be differentiated into fat cells by empirically determined hormonal cocktails. These in vitro systems appear to recapitulate most of the developmental events that take place in vivo and offer the advantage of synchronous differentiation and ease of manipulation. Studies of these cells, as well as cultured mouse embryonic fibroblasts (MEFs) derived from mice with various targeted gene ablations (and more recently, studies from intact animals) have revealed a transcriptional cascade in which the nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) plays a critical role. In cultured adipocytes and fat pads in vivo, PPAR␥ is both necessary and sufficient for adipogenesis (4, 33, 41). Other transcription factors have also been shown to play an important role in adipogenesis, including the CCAAT/enhancer binding proteins C/EBP␣, C/EBP␤, and C/EBP␦ and the basic helix-loop-helix protein SREBP1c (34). In 3T3-L1 cells exposed to a proadipog...

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by b-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold b 3 -adrenoceptor knockout mice (b 3 KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of b 3 KO mice, UCP1 expression levels were normal at 24°C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of b 3 KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that b 3 -adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.Keywords: adipose tissue; cold exposure; differentiation; knockout; uncoupling protein 1.Brown adipose tissue (BAT) and white adipose tissue (WAT) both play an important role in the control of energy balance in mammals. Indeed, BAT is involved in the control of body temperature and body weight via nonshivering and diet-induced thermogenesis [1,2] whereas the primary function of WAT is to store and release energy. BAT function is particularly important for thermoregulation in small mammals in which the surface to volume ratio is unfavorable [1]. Moreover, activation of BAT prevents obesity induced by overfeeding in rodents [2].Cold exposure, via activation of the sympathetic nervous system, induces heat production in BAT [3,4] and lipolysis in WAT [5]. The effects of norepinephrine released at the nerve endings are mediated in brown and white adipocytes mainly by the b 3 -adrenoceptor subtype [6].Brown and white adipocytes are usually located in distinct depots [4] and can be distinguished morphologically [7]: brown adipocytes contain multiple (multilocular) lipid droplets and numerous mitochondria whereas white adipocytes display a main (unilocular) lipid droplet and few mitochondria. Nevertheless, the morphological aspect alone is not sufficient to distinguish between these two cell types [8]. In fact, the only criterion is the specific expression in mitochondria of brown adipocytes, of the uncoupling protein-1 (UCP1), which uncouples the oxidative phosphorylations from electron transport, thus dissipating energy as heat [9].An observation made initially by Young et al.[10] was the presence of multilocular mitochondria-rich brown adipocytes in depots previously defined as typical WAT. The emergence of these ectopic cells was found to be induced by cold acclimation in WAT of rats [11,12] and of mice [13,14]. The new cells were found to be sympathetically innervated [15] and to remain present as long as a sympathetic nervous system stimulation persisted [16]. Several reports also showed that the administration of selective b 3 -adrenoceptor agonis...

Catecholamines are viewed as major stimulants of diet-and cold-induced thermogenesis and of-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, L L-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

OBJECTIVE-Recent observations indicate that the delivery of nitric oxide by endothelial nitric oxide synthase (eNOS) is not only critical for metabolic homeostasis, but could also be important for mitochondrial biogenesis, a key organelle for free fatty acid (FFA) oxidation and energy production. Because mice deficient for the gene of eNOS (eNOS Ϫ/Ϫ ) have increased triglycerides and FFA levels, in addition to hypertension and insulin resistance, we hypothesized that these knockout mice may have decreased energy expenditure and defective ␤-oxidation.RESEARCH DESIGN AND METHODS-Several markers of mitochondrial activity were assessed in C57BL/6J wild-type or eNOS Ϫ/Ϫ mice including the energy expenditure and oxygen consumption by indirect calorimetry, in vitro ␤-oxidation in isolated mitochondria from skeletal muscle, and expression of genes involved in fatty acid oxidation. RESULTS-eNOSϪ/Ϫ mice had markedly lower energy expenditure (Ϫ10%, P Ͻ 0.05) and oxygen consumption (Ϫ15%, P Ͻ 0.05) than control mice. This was associated with a roughly 30% decrease of the mitochondria content (P Ͻ 0.05) and, most importantly, with mitochondrial dysfunction, as evidenced by a markedly lower ␤-oxidation of subsarcolemmal mitochondria in skeletal muscle (Ϫ30%, P Ͻ 0.05). Finally, impaired mitochondrial ␤-oxidation was associated with a significant increase of the intramyocellular lipid content (30%, P Ͻ 0.05) in gastrocnemius muscle. CONCLUSIONS-These data indicate that elevated FFA and triglyceride in eNOSϪ/Ϫ mice result in defective mitochondrial ␤-oxidation in muscle cells. Diabetes

Uncoupling protein-3 (UCP3) is a mitochondrial innermembrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.

The mechanism of thermoregulatory uncoupling of respiration and phosphorylation in skeletal muscles has been studied. It is found that 24 h cold exposure results in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering of the membrane potential in isolated rat or mouse skeletal muscle mitochondria. The decrease in membrane potential is reversed by adding bovine serum albumin. Cold exposure is also found to sensitize the membrane potential to the uncoupling action of added fatty acid (laurate). After laurate addition, the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin increases in mitochondria from cold-treated rats or mice. Changes similar to those induced by cold can be initiated by the in vivo addition of thyroxine. Cold exposure does not affect energy coupling in liver mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive and -insensitive uncoupling is discussed.

Catecholamines are major stimulants of adipose tissue metabolism. Norepinephrine and epinephrine act through three subtypes of ␤-adrenoceptors (␤-AR) expressed in the adipocytes. The aim of this work was to study the mechanisms of lipid mobilization in ␤ 1 /␤ 2 /␤ 3 -AR triple-knockout (␤-less) mice. Glycerol and nonesterified fatty acids released from isolated adipocytes were measured as an index of lipolytic activity. There was no difference between the two genotypes for basal lipolysis and lipolytic response to corticotropin or to agents acting at the adenylyl cyclase and protein kinase A levels. The lipolytic response to norepinephrine and ␤-AR agonists was blunted in ␤-less mice. However, a residual low-affinity lipolytic effect was observed in the presence of catecholamines and ␤ 3 -AR agonists but not of ␤ 1 -or ␤ 2 -AR agonists. cAMP levels were increased by a ␤-AR agonist in white and brown adipocytes of ␤-less mice. The residual lipolytic effect was blocked by ␤-AR antagonists. It was mediated neither by ␣ 1 -or ␣ 2 -AR nor by dopaminergic, serotonergic, and histaminergic receptors. Bioinformatic analyses do not provide evidence for a fourth ␤-AR. We conclude that the residual lipolytic effect observed in ␤-less mice can be attributed to an unknown Gs-protein-coupled receptor with low affinity for catecholamines.